Two distinct regions of Mto1 are required for normal microtubule nucleation and efficient association with the -tubulin complex in vivo

JCS038414 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Anti-Mto1 immunofluorescence before (32°C) and after (0°C) cold-induced MT depolymerization. Single-label anti-Mto1 staining is shown in the black and white panels, and the same images with DAPI counterstaining are shown underneath. (A) Wild-type, mto1Δ and mto1 CM1-region mutants (strains KS515, KS1017, KS2007, KS2010, KS2013). (B) Wild-type, mto2Δ and mto1-334 cells (strains KS516, KS976, KS2272). As shown previously (Samejima et al., 2005), Mto1 does not redistribute to the nuclear envelope after cold treatment of mto2Δ cells, and the same is true for Mto1-334 in (wild-type) mto2⁺ cells. The mechanism of this redistribution is not known, nor is its physiological significance, as Mto2 is not required for Mto1 localization in normal exponentially growing cells (Samejima et al., 2005); for the purposes of this work it is important only to note the similar phenotypes of mto2Δ and mto1-334. Bar, 10 µm.

• Supplemental Table S1 - Adobe PDF

• Movie 1 -

  Movie 1. Time-lapse sequence of nmt81:GFP-Atb2 (green) and Sad1-dsRed (red) in wild-type cells (strain KS2863). Note extensive MT nucleation and dynamics during interphase, presence of cytoplasmic astral MTs during anaphase, and robust post-anaphase array of MTs at the end of mitosis. A few additional red spots are from background fluorescence. Images acquired every 20 seconds and played back at 10 frames per second; total time compression 200×.

• Movie 2 -

  Movie 2. Time-lapse sequence of nmt81:GFP-Atb2 (green) and Sad1-dsRed (red) in mto1Δ cells (strain KS3609). Note absence of cytoplasmic MT nucleation and unusual interphase MT dynamics, including bend-breakage events when MTs curve around cell tips. Note also that interactions between MTs and the SPB are very limited relative to mto2Δ mutants (see Movie 6). Note also that in the cell that completes mitosis by the end of the movie (middle right), nucleated MTs are restricted to within the nuclear volume (faintly visible). Images acquired every 20 seconds and played back at 10 frames per second; total time compression 200×.

• Movie 3 -

  Movie 3. Time-lapse sequence of nmt81:GFP-Atb2 (green) and Sad1-dsRed (red) in mto1-9A1 cells (strain KS3604). Note similarity to mto1Δ mutants (see Movie 2). A few cells contain supernumerary Sad1-dsRed spots, but in nearly all cases the identity of the true SPB is apparent. Images acquired every 20 seconds and played back at 10 frames per second; total time compression 200×.

• Movie 4 -

  Movie 4. Time-lapse sequence of nmt81:GFP-Atb2 (green) and Sad1-dsRed (red) in mto1-9A2 cells (strain KS3605). Note similarity to mto1Δ and mto1-9A1 mutants (see Movies 2, 3). A few cells contain supernumerary Sad1-dsRed spots. Images acquired every 20 seconds and played back at 10 frames per second; total time compression 200×.

• Movie 5 -

  Movie 5. Time-lapse sequence of nmt81:GFP-Atb2 (green) and Sad1-dsRed (red) in mto1-9A3 cells (strain KS3607). Note similarity to wild-type cells (see Movie 1) in both interphase and mitosis. A few cells contain supernumerary Sad1-dsRed spots. Images acquired every 20 seconds and played back at 10 frames per second; total time compression 200×.

• Movie 6 -

  Movie 6. Time-lapse sequence of nmt81:GFP-Atb2 (green) and Sad1-dsRed (red) in mto2Δ cells (strain KS2785). Note nucleation of interphase cytoplasmic MTs from the SPB, and occasional rocketing of SPBs back-and-forth in the cell, driven by MT polymerization pushing against cell tips. Note also presence of astral MTs in the single anaphase cell and weak post-anaphase array nucleation. A few cells contain supernumerary Sad1-dsRed spots. Images acquired every 20 seconds and played back at 10 frames per second; total time compression 200×.

• Movie 7 -

  Movie 7. Time-lapse sequence of nmt81:GFP-Atb2 (green) and Sad1-dsRed (red) in mto1-334 cells (strain KS3765). Note similarity to mto2Δ (see Movie 6), including interphase MT nucleation from the SPB and SPB rocketing, the presence of astral MTs in mitosis, and weak nucleation of post-anaphase array MTs. A few cells contain supernumerary Sad1-dsRed spots. Images acquired every 20 seconds and played back at 10 frames per second; total time compression 200×.